The NF-kB pathway promotes survival of cancer cells. My research in ovarian cancer began with characterizing the activation state and biological relevance of NF-kB in this disease. The NF-kB family of transcription factors is ubiquitously expressed. NF-kB signaling has been implicated in ovarian cancer, but the significance and mechanism of NF-kB signaling in ovarian cancer is unknown. There is precedent to propose that NF-kB is a critical signaling mechanism in cancer. I initially hypothesized that the NF-kB pathway is over-activated in ovarian cancers with more aggressive behavior. The NF-kB pathway was implicated in ovarian cancer proliferation and cytokine secretion in vitro, and contributed to chemoresistance of ovarian cancer cell lines. I therefore sought to determine the expression patterns and prognostic associations of NF-kB pathway proteins in primary ovarian cancer tissues. I demonstrated that overexpression of the NF-kB subunit p50 at diagnosis conveyed poor outcome in these patients. The biological relevance of NF-kB in ovarian cancer was established in my laboratory. Having demonstrated the coordinate presence of NF-kB machinery in ovarian cancers, I sought to modulate its activity. Inhibitors of NF-kB (IkBs) are tagged for degradation through the proteasome upon specific inducible phosphorylation by IkB kinases (IKKs). Therefore, targeted inhibition of IKKs could isolate NF-kB as a mechanism for ovarian cancer pathogenesis. A subset of ovarian cancer cell lines was affected by inhibition of IKKb in properties of growth, adhesion, invasion and cytokine secretion. I developed a gene expression signature of IKKb signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKb. This signature gave insight into the results of NF-kB in ovarian cancer, based on known functions of the ovarian cancer-specific target genes, and allowed me to probe established ovarian cancer databases in order to estimate the relative impact of NF-kB signaling on the survival of women with ovarian cancer. Higher NF-kB activity conveyed a worse outcome, suggesting that modulation of IKKb might benefit patients whose tumors showed elevated target gene expression. A key discovery from this work was the tissue specificity of NF-kB signaling. The 9-gene signature experimentally defined in ovarian cancer was completely different from the 11 genes I previously identified in multiple myeloma. The overall goal of this project is to dissect the molecular structure of NF-kB signaling in ovarian cancer, with the intent to develop biomarkers of dependence on NF-kB, and novel points of therapeutic intervention. We completed two shRNA library screens, one in combination with an inhibitor of IKKbeta, and another in combination with shRNA against IKKepsilon. These studies identified novel interactions between the NF-kB pathway in ovarian cancer. In combination with IKKbeta, we found caspase 8 to be cooperative in protecting the cells from necroptosis. This work continues with designing a translational strategy for improving clinical outcome of women with caspase 8-deficient cancers. We plan to target the necroptosis pathway therapeutically, to bypass the defective apoptosis that mediates resistance to standard chemotherapy regimens. In a related avenue, we continue to study NF-kB signaling in ovarian cancer tumor-initiating cells. We observed by immunofluorescence that classical NF-kB appeared to be active in only a sub-population of the cultures at any given time, based on the presence of NF-kB p65 in the nucleus. Elevated classical NF-kB signaling has been observed in tumor-initiating cells (TICs) of prostate, breast, and ovarian tumors, but there are limited studies examining alternative NF-kB signaling. Both classical and alternative signaling cascades are required for maintenance and promotion of breast cancer TICs. The classical and alternative NF-kB pathways can regulate each other and integrate with other signaling pathways for fine-tuning functional outputs. Thus, the diverse and complex roles of NF-kB suggest this transcription factor family regulates cellular functions in a context dependent manner and may be a key factor in maintaining heterogeneity. We are currently testing the hypothesis that classical and alternative NF-kB pathways support distinct subpopulations of ovarian cancer tumor-initiating spheroids, that collaborate to populate secondary tumors following chemotherapy. Our recent results suggest that NF-kB supports the TIC phenotype and functional outputs of NF-kB are responsible for proliferation, chemoresistance, differentiation, multipotency. A better understanding of the biology underlying NF-kB signaling in TIC and non-TIC populations of tumor cells will guide the design of more effective therapies to overcome chemoresistance, prevent relapse, and improve survival of women with ovarian cancer. From a therapeutic standpoint, we previously completed a phase 2 clinical trial using the SMAC mimetic birinapant - that can target NF-kB signaling via inhibition of IAP proteins - in women with relapsed and refractory ovarian cancer. There were no clinical responses. Therefore, through the NCI Major Opportunities, we completed a matrix drug screen testing 2000 compounds in combination with birinapant, in order to identify synergistic combinations to move to the clinic. We identified drug classes that met the criteria for synergy, and are in the process of testing these in mouse models. In addition, we initiated preclinical evaluation of another SMAC mimetic that targets both cIAP1 and XIAP, and will test whether this compound has better anti-cancer efficacy alone and in combination with chemotherapy and kinase inhibitors. Based on recent genomic analyses of cell lines, we now hypothesize that NF-kB activation contributes to resistance to the individual drugs; and that the SMAC mimetic blocks NF-kB activation and sets the molecular stage for apoptosis to ensue. Ongoing work will test this hypothesis using reporter assay, differential caspase activation (8 versus 9) and downstream cytokine measurements.